Synthesis, X-ray crystallography and antimicrobial activity of 2-cyanoguanidinophenytoin

The optimized synthesis of [5-oxo-4,4-diphenylimidazolidin-2-ylidene]cyanamide, which is known as 2-cyanoguanidinophenytoin (CNG-DPH) (3), and (imidazo[4,5-d]imidazole-2,5-diylidine)dicyanamide (4) has been reported in the present work. Furthermore, new Mannich bases derived from CNG-DPH were synthesized via its reaction with formaldehyde and using the corresponding amines, piperidine (base 5), and morpholine (base 6). Also, the antimicrobial activity and X-ray crystal structures for CNG-DPH and their Mannich bases were studied. The bases 3 and 6 crystallized in a monoclinic system; the crystal structure of 3 containing four molecules in the unit cell with a P21/c space group. The unit cell of 6 has eight molecules with a C2/c space group. The inter and intra hydrogen bond contacts packed and stabilized both of the structures. The morpholine ring of base 6 demonstrated a distinctive chair configuration. Mannich bases 5 and 6 showed promising antimicrobial effects. base 4 has a greater percentage for in vitro cytotoxicity (IC50) against normal cells, whereas 3 has the lowest ratio.

hydrochloric acid (Table 1, entry 1).Therefore, the reaction was optimized and examined to improve the yield of 3 and reduce the yield of compound 4 by changing various reaction conditions including the base concentration and the equivalent amount of cyanoguanidine.All results are summarized in Table 1.
The effect of the base on the reaction was screened with various molarities using different strong bases.While, using organic bases is not effective in reactions of cyanoguanidine 24 .The screening results indicated that sodium ethoxide and sodium methoxide are the most effective bases for this reaction (Table 1, entries 7-10).By increasing the base molarity to 4 mol equivalent, the yield of desired product 3 was improved to 80% and the yield of undesired product 4 was reduced to 5% (Table 1, entry 9), while by increasing number of moles more than 4 mol Table 1.Optimization of the synthesis of CNG-DPH 3 and (imidazo [4,5-d]imidazole-2,5-diylidine) dicyanamide 4. a Reported method 21 .b Formed as reported 21 but their yields are not detected.c  Figures 2 and 3 show molecular geometry of 3 and 6, from an Ortep perspective.The compounds crystallized in the monoclinic system possessing four molecules in the unit cell and P2 1 /c of 3 and eight molecules with C2/c space group for 6.The crystal structure of compound 3 is consistent with the same published structure 40 .
The main difference between the structures of 3 and 6 is the existence of a 4-methylmorpholine moiety in the structure of 6 (Figs. 2, 3).The molecular bond geometries indicated the connectivity of the atoms and were in agreement with the reported standard bond distances 41 .Calculations of the least-squares plane passing through the consisting atoms showed general planar configurations at 3 in every ring individually.However, all of the constituent parts exist in different planes.The phenyl rings have an angle 78.8° at 3 in agreement with the published structure (78.9°) 40 and 88.12° for 6 between the normal of their planes.The imidazole ring (N3/C11/N4/C9) in 6 showed non planar character, with maximum deviation from the plane at C9 (0.034 Å), and torsion angle N3/C11/N4/C9 (2.35°).Puckering investigations at 6-membered morpholine ring (O7/C22/C14/N6/C18/C26) of 6 indicated an obvious chair conformation (Fig. 4) 42 .
The structures are stabilized by the inter and intra molecular networks of hydrogen bond contacts, conformed parallel layers.The crystal packing is further stabilized by rings interaction (Cg-J), where Cg refers to the gravity center of a ring (J), and J identifies the ring number in a structure.It was found that the 5-Membered imidazole ring (Cg1) in compound 3, as well as the morpholine ring (Cg4) in compound 6, have gravity centers that interact with hydrogens (Tables 2, 3).The packing diagrams and intermolecular contacts of the compounds 3 and 6 are shown in Figs. 5 and 6, respectively.

Antimicrobial activity
The newly synthesized compounds were tested for their antimicrobial activity against the following microorganisms: two gram positive stains B. cereus, S. aureus P. and, two gram negative stains aeruginosa E. coli, S. typhi, and two fungal stains Candida albicans and Aspergillus niger.The preliminary screening of the investigated compounds was performed using Broth dilution method.The minimum inhibitory concentrations, were recorded accordingly.The antimicrobial activity of Mannich bases 5 and 6 was active against both Gram-positive, Gramnegative bacteria, Candida albicans and Aspergillus niger with different inhibition zone diameter range from 15.33 to 30.16 mm as mentioned in Table 4 and Fig. 7.
The table shows the inhibition zone (mm) of different microbial strains at a concentration of 10 mg/ml of different compounds.The inhibition zone is a measure of the antimicrobial activity of the tested compounds against the microbial strains.The results indicate that the tested compounds have varying degrees of antimicrobial activity against the different microbial strains.
Compounds 3 and 4 didn't show antimicrobial activity except E. coli with minimum inhibition zone 13.3 and 14.6 mm respectively: Rifampin demonstrated an inhibition zone diameter range from 11.3 to 34 mm against tested bacteria whereas fluconazole didn't demonstrate an inhibition zone against both C. albicans and A. niger.
It is important to note that the results of the study are limited to the specific concentrations and strains tested.Further studies are needed to determine the minimum inhibitory concentration (MIC) and the mechanism of action of the tested compounds against the different microbial strains.The results of the study may have implications for the development of new antimicrobial agents and the treatment of microbial infections.

Samples assessment
All the new molecules have been evaluated against normal cell lines (Vero cells), Fig. 8, which were obtained from American Type Culture Collection (ATCC, USA).The data presented in Table 5 indicate that compound

General procedure for the synthesis of Mannich bases 5 and 6
A mixture of cyanamide 3 (1 mmol, 0.27 g) and formaldehyde solution 27% (1.1 mmol, 0.12 ml) in 40 ml refluxing absolute ethanol was stirred for 15 min and then an appropriate secondary amine; piperidine and/ or morpholine (1 mmol) was added and refluxed for about 2 h (monitored with TLC).After completion of the reaction, the reaction mixture was cooled and the formed crystal 5 and/or 6, respectively was filtered off and used without further purification.

X-ray crystallography
Crystals from the studied compounds were carefully examined under an optical microscope in order to choose single crystals that were appropriate and free of imperfections like fractures and bubbles.The data gathered by an Enraf-Nonius 590 diffractometer with a Kappa CCD detector and a Mo-X-ray source at room temperature 43 .The measurements were performed at the X-ray Crystallography Laboratory of the National Research Centre.

Data analysis
The structures were solved and refined using the direct method with SHELXS97 and SIR92 software 44,45 implemented in maXus program suit 46 .The non-hydrogen atoms were refined with anisotropic displacement parameters 47 .The general-purpose crystallographic tool PLATON 48 was used for the structure analysis and presentation of the results.The molecular graphics were done using Mercury program 49 .Full crystallographic included in the supplementary (Tables S1-S3), also the structures deposited at Cambridge Crystallographic Data Centre with CCDC, deposition numbers 2207479 and 1881221 for bases 3 and 6, respectively 50 .

Antimicrobial activity
The antimicrobial activity of the tested compounds was determined by means of the agar diffusion method on Muller Hinton agar.The wells (8 mm diameter) were cut using a sterile cork borer on Muller Hinton agar (MHA, India) for bacteria and potato dextrose agar (India) for fungi.Twenty-four hours young culture of Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 10987, Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028 and 48 h young culture of Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404 were swabbed with a sterilized cotton swab on the surface of prepared Muller Hinton agar for bacteria and potato dextrose agar for fungi.One hundred microliters of dissolved compounds were loaded into each well and left for 2 h at 4 °C until the metabolite was diffused.Then the plates were incubated for 24 h at 37 °C for bacteria and 72 h at 28 °C for fungi.After incubation, the zone of inhibitions was measured and recorded 51,52 .

In vitro cytotoxicity assaying of the prepared compounds
The prepared compounds were screened for their anti-proliferative effect against normal Vero cells.Briefly, Vero cells were seeded at a density of 104 cells per well in a sterile 96-well microplate for 24 h in a 5% CO 2 incubator.In triplicate, different concentrations (31.25, 62.5,125, 250, 500 and 1000 µg/ml) of each prepared compound were applied to the cells and incubated for another 72 h.After discarding the medium containing compound, each well was filled with 200 ml MTT.The assay is primarily based on a biochemical reaction in which mitochondrial succinate dehydrogenase in viable cells converts the yellow tetrazolium bromide (MTT) to a purple formazan derivative.In general, the cells were cultured in RPMI-1640 medium with 10% fetal bovine serum.Penicillin (100 units/ml) and streptomycin (100 µg/ml) were added in a 5% CO2 incubator at 37 °C.Seeding of the cells was in a 96-well plate at 37 °C for 48 h under 5% CO2 and it was at a density of 1.0 × 10 4 cells/well.After the initial incubation, the cells were treated with the new derivatives at different concentrations and incubated for 24 h.Then we add 20 µl of MTT in a solution of 5 mg/ml concentration and incubate for 4 h.To dissolve the obtained purple formazan, 100 µl of DMSO was added into each well.Measuring and recording the colorimetric assay were carried out by a plate reader (EXL 800, USA) at an absorbance of 570 nm.The percentage of relative cell viability was calculated 53 .

Figure 3 .Figure 4 .
Figure 3.An ORTEP view of Mannich base 6 with atom-numbering.Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

Table 3 .Figure 5 .
Figure 5.A view of packing diagram for compound 3. Hydrogen bond contacts with dashed blue lines.

Figure 6 .
Figure 6.A view of packing diagram for compound 6.Hydrogen bond contacts with dashed blue lines.

Figure 9 .
Figure 9.Effect of compound 3 on Vero cell at different concentration.

Figure 10 .
Figure 10.Effect of compound 4 on Vero cell at different concentration.

Figure 11 .
Figure 11.Effect of compound 5 on Vero cell at different concentration.

Figure 12 .
Figure 12.Effect of compound 6 on Vero cell at different concentration.
Optimized condition.Significant values are in bold.

Table 4 .
Antimicrobial activity of compounds 3

Table 5 .
Determination of IC 50 of compounds 3-6 on Vero cell.